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PLoS Neglected Tropical Diseases Jan 2021Bovine tuberculosis (TB) caused by Mycobacterium bovis is a significant health threat to cattle and a zoonotic threat for humans in many developing countries. Rapid and...
Bovine tuberculosis (TB) caused by Mycobacterium bovis is a significant health threat to cattle and a zoonotic threat for humans in many developing countries. Rapid and accurate detection of M. bovis is fundamental for controlling the disease in animals and humans, and for the proper treatment of patients as one of the first-line anti-TB drug, pyrazinamide, is ineffective against M. bovis. Currently, there are no rapid, simplified and low-cost diagnostic methods that can be easily integrated for use in many developing countries. Here, we report the development of a loop-mediated isothermal amplification (LAMP) assay for specific identification of M. bovis by targeting the region of difference 4 (RD4), a 12.7 kb genomic region that is deleted solely in M. bovis. The assay's specificity was evaluated using 139 isolates comprising 65 M. bovis isolates, 40 M. tuberculosis isolates, seven M. tuberculosis complex reference strains, 22 non-tuberculous mycobacteria and five other bacteria. The established LAMP detected only M. bovis isolates as positive and no false positives were observed using the other mycobacteria and non-mycobacteria tested. Our LAMP assay detected as low as 10 copies of M. bovis genomic DNA within 40 minutes. The procedure of LAMP is simple with an incubation at a constant temperature. Results are observed with the naked eye by a color change, and there is no need for expensive equipment. The established LAMP can be used for the detection of M. bovis infections in cattle and humans in resource-limited areas.
Topics: Animals; Cattle; Humans; Molecular Diagnostic Techniques; Mycobacterium bovis; Nucleic Acid Amplification Techniques; Sensitivity and Specificity
PubMed: 33493196
DOI: 10.1371/journal.pntd.0008996 -
Molecules (Basel, Switzerland) Sep 2022Bacillus Calmette-Guérin polysaccharide and nucleic acid (BCG-PSN), extracted from , is an immunoregulatory medicine commonly used in clinic. However, the structural...
Bacillus Calmette-Guérin polysaccharide and nucleic acid (BCG-PSN), extracted from , is an immunoregulatory medicine commonly used in clinic. However, the structural characteristics and potential pharmacological efficacy of the polysaccharides from BCG-PSN remain unclear. Herein, two polysaccharides (BCG-1 and BCG-2) were purified and their structures were characterized. Monosaccharide composition analysis combined with methylation analysis and NMR data indicated that BCG-1 and BCG-2 were an α-D-(1→4)-mannan with (1→2)-linked branches, and an α-D-(1→4)-glucan with (1→6)-linked branches, respectively. Herein, the mannan from BCG-PSN was first reported. Bioactivity assays showed that BCG-1 and BCG-2 dose-dependently and potently increased the production of inflammatory mediators (NO, TNF-α, IL-6, IL-1β, and IL-10), as well as their mRNA expressions in RAW264.7 cells; both have similar or stronger effects compared with BCG-PSN injection. These data suggest that BCG-1 and BCG-2 are very likely the active ingredients of BCG-PSN.
Topics: Adjuvants, Immunologic; BCG Vaccine; Mannans; Mycobacterium bovis; Polysaccharides
PubMed: 36080458
DOI: 10.3390/molecules27175691 -
Applied and Environmental Microbiology Apr 2005PCR primers specific for the Mycobacterium tuberculosis complex were used to detect the presence of Mycobacterium bovis BCG (Pasteur) in soil microcosms and...
PCR primers specific for the Mycobacterium tuberculosis complex were used to detect the presence of Mycobacterium bovis BCG (Pasteur) in soil microcosms and Mycobacterium bovis in environmental samples taken from a farm in Ireland with a history of bovine tuberculosis. M. bovis genes were detected in soil at 4 and 21 months after possible contamination. Gene levels were found in the range of 1 x 10(3) to 3.6 x 10(3) gene copies g of soil(-1), depending on the sampling area. Areas around badger setts had the highest levels of detectable genes and were shown to have the highest levels of gene persistence. M. bovis-specific 16S rRNA sequences were detected, providing evidence of the presence of viable cells in Irish soils. Studies of DNA turnover in soil microcosms proved that dead cells of M. bovis BCG did not persist beyond 10 days. Further microcosm experiments revealed that M. bovis BCG survival was optimal at 37 degrees C with moist soil (-20 kPa; 30% [vol/wt]). This study provides clear evidence that M. bovis can persist in the farm environment outside of its hosts and that climatic factors influence survival rates.
Topics: Agriculture; Animals; BCG Vaccine; Bacterial Proteins; Cattle; DNA Primers; DNA, Bacterial; Ireland; Mycobacterium bovis; Polymerase Chain Reaction; RNA, Bacterial; RNA, Ribosomal, 16S; Soil Microbiology; Species Specificity; Tuberculosis, Bovine
PubMed: 15812024
DOI: 10.1128/AEM.71.4.1946-1952.2005 -
PloS One 2016Mycobacterium bovis is the causative agent of bovine tuberculosis (BTB), the pathogen responsible for serious economic impact on the livestock sector. In order to obtain...
Mycobacterium bovis is the causative agent of bovine tuberculosis (BTB), the pathogen responsible for serious economic impact on the livestock sector. In order to obtain data on isolated M. bovis strains and assist in the control and eradication program for BTB, a cross sectional descriptive molecular epidemiology study in the Brazilian Midwest was conducted. Through spoligotyping and 24-loci MIRU-VNTR methods, 37 clinical isolates of M. bovis circulating in the region were analyzed, 10 isolated from the state of Mato Grosso, 12 from the state of Mato Grosso do Sul and 15 from the state of Goiás. The spoligotyping analysis identified 10 distinct M. bovis profiles (SB0121 n = 14, SB0295 n = 6, SB0140 n = 6, SB0881 n = 3, SB1144 n = 2, SB1145 n = 2, SB0134 n = 1, SB1050 n = 1, SB1055 n = 1, SB1136 n = 1) grouped in six clusters and four orphan patterns. The MIRU-VNTR 24-loci grouped the same isolates in six clusters and 22 unique orphan patterns, showing higher discriminatory power than spoligotyping. When associating the results of both techniques, the isolates were grouped in five clusters and 24 unique M. bovis profiles. Among the 24-loci MIRU-VNTR evaluated, two, ETR-A and QUB 11b loci, showed high discriminatory ability (h = ≥ 0.50), while MIRU 16, MIRU 27, ETR-B, ETR-C, Mtub21 and QUB 26 loci showed moderate ability (h = 0.33 or h = 0.49) and were the most effective in evaluating the genotypic similarities among the clinical M. bovis isolate samples. Herein, the 29 patterns found amongst the 37 isolates of M. bovis circulating in the Brazilian Midwest can be due to the animal movement between regions, municipalities and farms, thus causing the spread of various M. bovis strains in herds from Midwest Brazil.
Topics: Animals; Cattle; Cluster Analysis; Genes, Bacterial; Mycobacterium bovis
PubMed: 27631383
DOI: 10.1371/journal.pone.0162459 -
IUBMB Life Mar 2022Posttranslational modifications (PTMs) could influence many aspects of protein behavior and function in organisms. Protein glycosylation is one of the major PTMs...
Posttranslational modifications (PTMs) could influence many aspects of protein behavior and function in organisms. Protein glycosylation is one of the major PTMs observed in bacteria, which is crucial for functional regulations of many prokaryotic and eukaryotic organisms. Mycobacterium bovis bacillus Calmette-Guérin (BCG) vaccine has been recognized as an indispensable tool in the global fight against tuberculosis (TB) worldwide over several decades. Nevertheless, analysis of glycoprotein profiles of BCG has not been clearly investigated. In this study, we performed O-mannosylated protein analysis in BCG bacteria using gel-based and gel-free approaches. In total, 1,670 hexosylated peptides derived from 754 mannosylated proteins were identified. Furthermore, 20 novel protein products supported by 78 unique peptides not annotated in the BCG database were detected. Additionally, the translational start sites of 384 proteins were confirmed, and 78 proteins were validated through the extension of translational start sites based on N-terminus-derived peptides. The bioinformatic analysis of the O-mannosylated proteins was performed and the expression profiles of four randomly selected proteins were validated through Western blotting. A number of proteins involved in metabolic pathways, including the tricarboxylic acid cycle, glycolysis, oxidative phosphorylation, and two-component system, are discussed. Taken together, these results offer the first O-mannosylated protein analysis of a member of mycobacteria reported to date by using complementary gel-based and gel-free approaches. Some of the proteins identified in this study have important roles involved in metabolic pathways, which could provide insight into the immune molecular mechanisms of this recognized vaccine strain.
Topics: BCG Vaccine; Glycosylation; Humans; Mycobacterium bovis; Proteomics; Tuberculosis
PubMed: 34773437
DOI: 10.1002/iub.2578 -
Journal of Bacteriology Aug 2016Phthiocerol dimycocerosates (PDIM) are a group of cell surface-associated apolar lipids of Mycobacterium tuberculosis and closely related mycobacteria, such as...
UNLABELLED
Phthiocerol dimycocerosates (PDIM) are a group of cell surface-associated apolar lipids of Mycobacterium tuberculosis and closely related mycobacteria, such as Mycobacterium bovis and Mycobacterium leprae A characteristic methoxy group of these lipids is generated from the methylation of a hydroxyl group of the direct precursors, the phthiotriols. The precursors arise from the reduction of phthiodiolones, the keto intermediates, by a ketoreductase. The putative phthiodiolone ketoreductase (PKR) is encoded by Rv2951c in M. tuberculosis and BCG_2972c in M. bovis BCG, and these open reading frames (ORFs) encode identical amino acid sequences. We investigated the cofactor requirement of the BCG_2972c protein. A comparative analysis based on the crystallographic structures of similar enzymes identified structural elements for binding of coenzyme F420 and hydrophobic phthiodiolones in PKR. Coenzyme F420 is a deazaflavin coenzyme that serves several key functions in pathogenic and nonpathogenic mycobacteria. We found that an M. bovis BCG mutant lacking F420-dependent glucose-6-phosphate dehydrogenase (Fgd), which generates F420H2 (glucose-6-phosphate + F420 → 6-phosphogluconate + F420H2), was devoid of phthiocerols and accumulated phthiodiolones. When the mutant was provided with F420H2, a broken-cell slurry of the mutant converted accumulated phthiodiolones to phthiocerols; F420H2 was generated in situ from F420 and glucose-6-phosphate by the action of Fgd. Thus, the reaction mixture was competent in reducing phthiodiolones to phthiotriols (phthiodiolones + F420H2 → phthiotriols + F420), which were then methylated to phthiocerols. These results established the mycobacterial phthiodiolone ketoreductase as an F420H2-dependent enzyme (fPKR). A phylogenetic analysis of close homologs of fPKR revealed potential F420-dependent lipid-modifying enzymes in a broad range of mycobacteria.
IMPORTANCE
Mycobacterium tuberculosis is the causative agent of tuberculosis, and phthiocerol dimycocerosates (PDIM) protect this pathogen from the early innate immune response of an infected host. Thus, the PDIM synthesis system is a potential target for the development of effective treatments for tuberculosis. The current study shows that a PDIM synthesis enzyme is dependent on the coenzyme F420 F420 is universally present in mycobacteria and absent in humans. This finding expands the number of experimentally validated F420-dependent enzymes in M. tuberculosis to six, each of which helps the pathogen to evade killing by the host immune system, and one of which activates an antituberculosis drug, PA-824. This work also has relevance to leprosy, since similar waxy lipids are found in Mycobacterium leprae.
Topics: Amino Acid Sequence; Bacterial Proteins; Carbohydrate Dehydrogenases; Gene Expression Regulation, Bacterial; Lipids; Mycobacterium bovis; Mycobacterium tuberculosis; Phylogeny
PubMed: 27185825
DOI: 10.1128/JB.01035-15 -
Emerging Infectious Diseases Jun 2015Panama remains free of zoonotic tuberculosis caused by Mycobacterium bovis. However, DNA fingerprinting of 7 M. bovis isolates from a 2013 bovine tuberculosis outbreak...
Panama remains free of zoonotic tuberculosis caused by Mycobacterium bovis. However, DNA fingerprinting of 7 M. bovis isolates from a 2013 bovine tuberculosis outbreak indicated minimal homology with strains previously circulating in Panama. M. bovis dispersion into Panama highlights the need for enhanced genotype testing to track zoonotic infections.
Topics: Animals; Cattle; Genotype; History, 21st Century; Minisatellite Repeats; Molecular Typing; Mycobacterium bovis; Panama; Phylogeny; Polymorphism, Genetic; Tuberculosis, Bovine
PubMed: 25988479
DOI: 10.3201/eid2106.141821 -
ELife Dec 2019Quantifying pathogen transmission in multi-host systems is difficult, as exemplified in bovine tuberculosis (bTB) systems, but is crucial for control. The agent of bTB,...
Quantifying pathogen transmission in multi-host systems is difficult, as exemplified in bovine tuberculosis (bTB) systems, but is crucial for control. The agent of bTB, , persists in cattle populations worldwide, often where potential wildlife reservoirs exist. However, the relative contribution of different host species to bTB persistence is generally unknown. In Britain, the role of badgers in infection persistence in cattle is highly contentious, despite decades of research and control efforts. We applied Bayesian phylogenetic and machine-learning approaches to bacterial genome data to quantify the roles of badgers and cattle in infection dynamics in the presence of data biases. Our results suggest that transmission occurs more frequently from badgers to cattle than (10.4x in the most likely model) and that within-species transmission occurs at higher rates than between-species transmission for both. If representative, our results suggest that control operations should target both cattle and badgers.
Topics: Animals; Animals, Wild; Bayes Theorem; Cattle; Disease Reservoirs; Genome, Bacterial; Genomics; Host-Pathogen Interactions; Mustelidae; Mycobacterium bovis; Phylogeny; Tuberculosis, Bovine
PubMed: 31843054
DOI: 10.7554/eLife.45833 -
The European Respiratory Journal Nov 2016
Review
Topics: Adult; Animals; Antitubercular Agents; Female; Humans; Male; Middle Aged; Mycobacterium bovis; Treatment Outcome; Tuberculosis
PubMed: 27540021
DOI: 10.1183/13993003.00629-2016 -
Journal of Microbiology, Immunology,... Dec 2012Mycobacterium bovis frequently infects wild and farm deer species with tuberculosis. This study investigated mycobacterial infection in two native deer species Cervus... (Comparative Study)
Comparative Study
BACKGROUND/PURPOSE
Mycobacterium bovis frequently infects wild and farm deer species with tuberculosis. This study investigated mycobacterial infection in two native deer species Cervus unicolor swinhoei (Formosan Sambar, Sambar) and C. nippon taiouanus (Formasan Sika, Sika).
METHODS
Based on different sampling sources of 19 intradermal tuberculin test (ITT) Sambar, mycobacterial infection and/or species were detected by acid-fast stain, duplex polymerase chain reaction (PCR) and multiplex nested PCR (mnPCR) methods, traditional mycobacterial culture and gross lesion. Blood samples of 167 Sambar deer and 147 Sika deer were then tested by duplex PCR and mnPCR methods to investigate the prevalence of mycobacterial infection. Sequence variations of these mycobacterial species were analyzed as well.
RESULTS
Duplex PCR and mnPCR assays could differentiate between MTBC (M. bovis and M. tuberculosis) and M. avium, as well as between M. bovis and M. tuberculosis, respectively. These PCR methods showed a higher detection rate than traditional culture and matched the gross lesions examined in 19 ITT-examined Sambar. Therefore, the mycobacterial infection in blood samples of 314 deer samples was detected using these PCR methods. Duplex PCR and mnPCR showed an identical prevalence of 16.1% in Sambar and 8.2% in Sika and a significant difference in prevalence between these two deer species. M. bovis and M. tuberculosis were the species detected in feedlot Sambar and Sika. M. tuberculosis was found only and first in Sambar fed in central Taiwan. Sequence analysis revealed diverse genetic variations in M. bovis and M. tuberculosis associated with deer subspecies.
CONCLUSION
Multiplex PCR methods were established, and M. bovis and M. tuberculosis were identified in feedlot deer in Taiwan. Sequence variations indicated diverse sources of both mycobacterial species.
Topics: Animals; Bacteriological Techniques; Culture Media; Deer; Mycobacterium bovis; Mycobacterium tuberculosis; Polymerase Chain Reaction; Prevalence; Sensitivity and Specificity; Sequence Analysis, DNA; Species Specificity; Taiwan; Tuberculin Test; Tuberculosis
PubMed: 22578646
DOI: 10.1016/j.jmii.2011.12.022